Forensic and Clinical Toxicology Studies Focusing on Drug Analysis in Hair and Other Biological Matrices.

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Abstract

Clinical and forensic toxicology analysts rely heavily in their daily tests on the
analysis of the conventional samples (blood and urine). However, these specimens
are limited in the time scale they reflect with regard to drug intake history and also in
terms of drug stability within the matrices. Alternative matrices such as hair, oral
fluids and dried blood spots (DBS) provide new horizons and new opportunities.
Drugs incorporated within hair are very stable. Hair also provides a very long
detection window, for at least one year, if not a lot longer. Oral fluids on the other
hand are non-intrusive, easy to collect and much cleaner sample matrix than blood
or urine. DBS also offer great drug stability, are easy to collect, faster to analyse and
suitable for automated analysis. However, a number of studies are needed to
assess the limits of these alternative samples in terms of the correlation of their
results with the results of conventional samples and with regard to drug stability.
Such studies will enable a more reliable and confident interpretation of results
obtained from these matrices especially for medico-legal purposes.
The main aims of this research were: to develop and validate analytical methods for
detection and quantitation of drugs of use and abuse in hair, oral fluids, blood and
DBS samples, to investigate the correlation between dose and drug concentration in
hair, blood and oral fluids after controlled chronic drug administration, to investigate
the stability of anti-psychotic drugs in DBS (from patients) stored under different
conditions and the effect of addition of preservative, and to investigate the alcohol
intake prevalence among Kuwaiti drug addicts and correlate these results with selfreported
intake.
As the majority of drugs were basic, an extraction method based on methanolic
incubation was developed for detection of basic/weak basic drugs in hair. It was
compared to alkaline digestion (with NaOH) followed by liquid-liquid extraction
(LLE). Detection was achieved by LC-MS/MS (Sciex2000) after separation on a C18
column. When applying both methods on positive authentic hair samples the results
showed that the methanolic method was capable of extracting most basic drugs in
hair but only partially, while the alkaline digestion method was found to degrade
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some unstable drugs like sulpiride, but was capable of fully extracting the alkaline
stable drugs such as quetiapine.
After development and validation of the LLE-LC-MS(Exactive) method for the
analysis of anti-psychotics in blood, oral fluids and hair, an investigation was carried
out on the correlation pattern between trough concentrations in those three
matrices. The most significant correlation coefficients (r) found were those between
blood and hair concentrations, procyclidine r=0.83 (18 subjects p=<0.001),
risperidone r=0.96 (14 subjects p=<0.001), haloperidol r=0.90 (10 subjects
p=<0.001), OH-risperidone r=0.24 (13 subjects p=>0.44), quetiapine r=0.28 (14
subjects p=>0.33) and chlorprothixene r=0.32 (13 subjects p=>0.32). Among the
interesting results was the strong correlation found between drugs half-lives and the
mean ratio of hair concentration/dose (r=0.96, p=<0.003).
The stability of anti-pyschotics in DBS from patients’ samples was assessed by
storing them at four different temperatures (25, 4, -20 and -80°C) with and without
prior impregnation of the DBS cards with sodium fluoride. After development and
validation of the LLE-LC-MS method, samples were analysed at days 0, 45, 90 and
180. Results showed good stability of all the compounds (procyclidine, quetiapine,
risperidone, OH-risperidone, chlorprothixene and haloperidol) in all the different
storage conditions and no significant increase or decrease in drug concentrations
with sodium fluoride impregnation.
Finally, after trials with five different HPLC columns, two SPE cartridges, two LLE
extraction procedures and two mass spectrometer instruments, a method was
developed and validated for the detection and quantitation of alcohol’s minor and
specific metabolite in hair, ethyl glucuronide (EtG). The method has a limit of
detection (LOD) of 3pg/mg and lower limit of quantitation (LLOQ) of 9pg/mg. This
method was applied to 59 hair samples from patients at a general addiction centre
and alcohol prevalence was investigated and its correlation with self-reported use
was investigated.